Two structures of IgGs constituting the immunoglobulins (antibody molecules) of camelids are known to exist: one a heterotetramer having heavy chains and light chains, and the other consisting of a heavy-chain dimer (Isr. J. Vet. Med. 43(3), 198 (1987); Hamers-Casterman et al., Nature, 363, 446 (1993)). The tetrameric structure is a common characteristic of IgGs among humans and most animals. On the other hand, the latter IgG having a heavy-chain dimer structure is considered characteristic of camelids. The IgGs consisting of a heavy chain dimer of camelids does not accidentally result from pathologic conditions.
Immunoglobulins lacking light chains have been found in Camelus bactrianus and Camelus dromedarius, which are Asian and African camelids, as well as in all species of South American camelids. South American camelids include Lama pacos, Lama glama, and Lama vicugna. The molecular weight of dimeric IgG differs depending on the animal species. The molecular weight of heavy chains constituting these immunoglobulins is approximately 43 kDa to approximately 47 kDa, and normally are 45 kDa.
Another characteristic of the heavy-chain dimer IgG is that this antibody lacks the first domain of the constant region called CH1 according to the definition by Roitt et al. Furthermore, the hinge region has an amino acid sequence different from that of a normal heterotetrameric antibody (heavy chains+light chains). Based on the differences in the amino acid sequences, the IgGs of dromedaries are classified as follows (Hamers-Casterman et al., Nature 363, 446 (1993)):    IgG2: comprising a long hinge sequence (SEQ ID NO: 8);    IgG3: comprising a short hinge sequence (SEQ ID NO: 9); and    IgG1: heterotetrameric antibody.
Since the VH region of a heavy chain dimer IgG does not have to make hydrophobic interactions with a light chain, the region in the heavy chain that normally contacts a light chain is mutated to hydrophilic amino acid residues. Due to structural differences compared to VHs of normal heterotetrameric IgGs, VH domains of the heavy-chain dimer IgGs are called Variable domain of the heavy-chain of heavy-chain antibody (VHH).
VHH has excellent solubility due to its hydrophilic amino acid residues. Amino acid substitutions are scattered throughout the primary structure (amino acid sequence) of VHH. Additionally, these hydrophilic amino acid residues form a cluster in the space of the tertiary structure of VH corresponding to the site that interacts with the VL domain. Herein, the aforementioned space of the tertiary structure is specifically called former VL side. These amino acid substitutions are, for example, V37F or V37Y, G44E, L45R or L45C, and W47 are also mostly substituted with Gly. Such substitutions increase the hydrophilicity of the former VL side of VHH.
Therefore, the solubility of VHH is much higher than that of VH isolated and purified from humans or mice (single domain antibody; Ward et al., Nature, 341, 544 (1989)). VHH can be easily concentrated to 10 mg/mL in ordinary buffer solutions without any signs of aggregation. This concentration corresponds to, for example, approximately 100 times the solubility of mouse VH.
Furthermore, VHHs derived from camels and llamas have very high thermostability compared to mouse heterotetrameric antibodies. The use of VHH derived from these species can provide, for example, molecules that maintain their antigen binding ability even at 90° C. (van der Linden et al., Biochim. Biophys. Acta 1431 (1), 37 (1999))
The diversity of antibody repertoire of camelids is determined by the complementary determining regions (CDR) 1, 2, and 3 in the VH or VHH regions. Possession of three CDRs is in common with the IgGs of other animal species. However, the CDR3 in the camel VHH region is characterized by its relatively long length averaging 16 amino acids (Muyldermans et al., Protein Engineering 7(9), 1129 (1994)). For example, compared to the CDR3 of mouse VH having an average of 9 amino acids, the CDR3 of camel IgG is very long.
Most antigen binding sites of structurally known heavy chains+light chains heterotetrameric antibodies are known to form antigen-binding surfaces such as grooves, cavities, and flat areas (Webster et al., Current Opinion in Structural Biology 4, 23 (1994)). Therefore, when an epitope of a substance to be bound also forms a groove or a cavity, the antigen-binding site of the antibody may not bind well. For example, in proteins including enzymes, catalytic or functional residue, or toxic region is often located at the interior of a cleft. This structure facilitates extremely specific interactions of enzyme substrates and receptors with proteins. However, structures such as cavities and clefts are difficult for heterotetrameric antibodies to recognize, and therefore, they do not have high immunogenicity.
In contrast, there are reports that VHHs of camelids can specifically recognize clefts and cavities due to their characteristic structure described above. For example, in an experiment where antibodies were isolated from peripheral blood of camels immunized with an enzyme as the antigen, antibodies that seal the enzyme active center existed only among the camel IgG2 and IgG3, and not in IgG1 (Lauwereys et al., EMBO J. 17(13), 3512 (1998)). Furthermore, VHH having lysozyme activity inhibitory effect was isolated by the phage display method from a library derived from camels immunized with lysozyme (Arbabi Ghahroudi et al., FEBS Letters 414, 521 (1997)). The structure of the isolated VHH in complex with lysozyme was elucidated by X-ray crystallographic analysis (Desmyter et al., Nature Structural Biology 9, 803 (1996)). The results showed that in IgG2 or IgG3 of the camel antibody, the CDR3 region having a long protrusion is inserted and bound to the substrate-binding site of the enzyme such that the active center is sealed to cause competitive inhibition.
Industrially useful characteristics can be found in VHHs derived from camelids such as high solubility and the possible existence of novel activity that cannot be expected from tetrameric IgGs. To obtain an antibody variable region, an animal must be immunized with an antigen of interest to separate an antibody. However, such a classical method involves problems such as the need to purify large amounts of antigens and generation of non-specific antibodies. Accordingly, as a method for more easily obtaining an antibody variable region, a screening method using an rgdp library has been proposed. The phrase “rgdp library” refers to a library consisting of a genetic display package wherein genes encoding substances with binding affinity, such as antibody variable regions, display their expression products. A representative example of an rgdp library includes a phage library that displays antibody variable regions.
The method of obtaining antibodies using a phage library displaying antibody variable regions is being noticed as a novel method for obtaining antibodies that succeed to labor-intensive, classical methods of antibody production. The present inventors have also constructed novel antibody libraries that allow efficient acquisition of antibody variable regions, and have already filed a patent application (WO 01/62907). It would also be useful for VHHs to construct a library that allows to freely select a VHH with a binding affinity towards an arbitrary antigen from the library. However, several problems have been pointed out in the construction of camelid VHH libraries.
Another characteristic of the structure of camelid VHH is that it often contains a cysteine residue in the CDR3 in addition to cysteines normally existing at positions 22 and 92 of the variable region. The cysteine residues in CDR3 are considered to form disulfide bonds with other cysteines in the vicinity of CDR1 or CDR2 (Muyldermans et al., Protein Engineering 7(9), 1129 (1994); Muyldermans et al., J. Mol. Recognit. 12, 131 (1999)). CDR1 and CDR2 are determined by the germline V gene. They play important roles together with CDR3 in antigen binding (Desmyter et al., Nature Structural Biology, 9, 803 (1996); Structure 7(4), 361-370 (1999); Spinelli et al., J. Mol. Biol. 311(1), 123 (2001)). In general, the term “germline” refers to chromosomal genes maintained in germ cells, i.e., chromosomal genes that have not undergone rearrangement. Herein, among the chromosomal genes, particularly the region that constitutes the antibody gene is referred to as germline.
Recently, germlines of dromedaries and llamas belonging to Camelidiae were studied. As a result, IgGs of dromedaries and llamas were classified according to the length of CDR2 and cysteine positions in the V region (Nguyen et al., EMBO J. 19(5), 921 (2000); Harmsen et al., Mol. Immunol. 37, 579 (2000)).
However, it has been noted that the antibody genes of the entire germline of a dromedary cannot be considered as sufficiently covered by hitherto obtained antibody genes. For example, the concentration of the classification of cDNA nucleotide sequences of the antibodies obtained so far on particular classes reveal that the germlines from which these antibodies were derived had been biased (Nguyen et al., EMBO J. 19 (5), 921 (2000)). Furthermore, methodologically, they were considered to imply problems as follows. Specifically, known libraries were constructed using only one type of primer as the N-terminal primer. Therefore, due to problems of specificity, some germlines from which the VHH genes are derived might have leaked, or the amplification products might have been biased (Arbabi Ghahroudi et al., FEBS Letters 414, 521 (1997)).
A library having biased constituent genes is poor in repertoire. Therefore, screening of such a library may not yield antibodies against an antigen of interest. This may be the reason why antibodies inhibiting or promoting an enzyme activity could not be obtained from phage libraries derived from non-immunized camels.
Prior art proposes methods to immunize camels or llamas in advance with a sufficient amount of antigen in order to obtain the variable region of immunoglobulin heavy chains of camels or llamas (Published Japanese Translation of International Publication No. Hei 11-503918; Lauwereys et al., EMBO J. 17(13), 3512 (1998); Arbabi Ghahroudi et al., FEBS Letters 414, 521 (1997)). This method utilizes the phenomenon that the immune systems of camels and llamas mature their own heavy chain antibodies in vivo (Published Japanese Translation of International Publication No. 2000-515002; J. Immuno. Methods 240, 185 (2000)). Based on this method, antibodies that recognize lysozyme, tetanus toxoid, carbonic anhydrase, amylase, RNaseA, azo dye and such have been obtained.
However, the need of immunological sensitization in this method imposes various restrictions such as those described below:
necessity of immunological sensitization period;
toxic influence of immunogens on camelids;
difficulty in obtaining antibodies against substances with low immunogenicity; and
necessity of relatively large amounts of antigens for immunological sensitization.
Furthermore, to avoid the problems of immunological sensitization, a method comprising the following steps was proposed (Published Japanese Translation of International Publication No. 2000-515002):    1) randomly selecting camelid heavy-chain antibodies;    2) isolating coding sequences and cloning them into phage display vectors;    3) modifying those coding sequences in at least one codon by random substitution;    4) constructing a library of the randomly mutated coding sequences in the phage display vectors;    5) expressing the coding sequences in phages transfected with those vectors; and    6) subsequently, sorting the phages with immobilized antigen to select recognition molecules specific to the antigen.
As an alternate solution, a method using the framework of camel antibody has also been suggested. According to this method, camel antibodies are reconstructed by incorporating CDR1, CDR2, and CDR3 of VHH and VH into the camel antibody framework. This method applies the method developed for humanizing mouse VH. The loops of each CDR can be mutated randomly to enlarge the repertoire size. As a result, the affinity and specificity of the antibodies can be controlled (Published Japanese Translation of International Publication No. 2000-515002).
All of these solutions are based on the principle of aiming to attain diversity by introducing artificial mutations into the coding sequences. However, most of these attempts require a great deal of effort and complicated procedures to introduce the mutations, and takes a long time. Furthermore, many of such attempts accompanied inefficiency of producing overhigh inactive antibodies along with the production of active antibodies.
A conceivable alternative method involves constructing a phage library by incorporating VHH genes obtained from tissues and blood of non-immunized camels into phage display vectors, and selecting recognition molecules specific to an antigen by selecting phages that have binding ability from the phage library using the immobilized antigen. However, this method had been contemplated to only yield VHHs against substances with sufficient immunogenicity. Therefore, methods using phage libraries incorporating VHH genes had not been sufficiently studied (Published Japanese Translation of International Publication No. 2000-515002). Accordingly, non-immunized camel-derived VHH antibody phage libraries comprising a repertoire diverse enough to yield antibodies inhibiting or promoting enzyme activities had not existed.